| Objective Using genetic engineering means to transfect double gene eukaryotic plasmid vectors pCDNA3.1-NGF-IRES-BMP2 into rat BMSCs,investigate the effects of transfected the double gene BMP2 and NGF and inducd osteogenesis in rat bone marrow stromal cells. Method BMSCs were cultivated by whole bone marrow culture method in three months SD rats,after cervical dislocation executed,Cultivation by whole bone marrow culture method in rat BMSCs and stability batches. Cell growth forms were observed by inverted contrast microscope. Using flow cytometry instrument to detect rat BMSCs surface marker CD31, CD90. The cells were identified by flow cytometry. Attained the three generation of rats were divided into five groups, single gene pCDNA3.1-NGF transfection model( A group),single gene pCDNA3.1-BMP2 transfection model(B group), double gene p CDNA3.1-NGF-IRES-BMP2 transfection model(C group), Empty plasmid in the control group(D group),control group(E group)。Each group was transfected by Lipofectamine2000 mediate. Target gene protein expression was tested by western-blot. Type I collagen protein expression levels were detected by immumohistochemical staining. The gray value of each group was measured by using BioRad gel image analysis system. Calcium nodules numbers were stained by Alizarin red and counted. The data were statistically analyzed. Result The cells were isolated and cultured. CD90 positive and CD31 negative of in bone marrow stromal cells surface markers were detectd by flow cytometry. After transfection, the target protein expression levels and calcium nodules of subsequent analysis in double gene transfection group were higher than that of single group. There is a difference in statistical analysis(p < 0.05). Conclusion Double gene pCDNA3.1-NGF-IRES-BMP2 transfection group in BMSCs can express two kinds of target proteins at the same time, and the osteoblast differentiation of BMSCs can be induced, and promote it osteogenesis. |
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